This invention relates to a method and apparatus for analyzing chemical compositions and more particularly to a method and system for combining free zone electrophoretic separation of a mixture sample with electrospraying to interface with on-line detection or off-line collection apparatus.
Numerous systems employed in the separation and analysis of analytes are known in the prior art. However, these prior art systems are not necessarily broadly applicable to the separation and/or analysis of analytes which comprise complex materials, or high molecular weight, nonvolatile, and highly polar compounds.
One known method for separation of analyte mixtures, free zone electrophoresis in small diameter capillaries or capillary zone electrophoresis (CZE), is used for a wide variety of analyses including high resolution separations of amino acids, peptides, proteins and complex salt mixtures. CZE employs a capillary with a electric field gradient to separate the analyte constituents, particularly ions, by difference in electrophoretic mobilities in addition to electroosmotic flow in the capillary. The electroosmotic flow results when an electrical double layer of ions forms at the capillary surface, and an electrical field is imposed lengthwise along the capillary. The field causes the ions to migrate towards the oppositely charged electrode at rates determined by the electrophoretic mobility of each analyte. In the resulting bulk electroosmotic flow, positively charged ions, neutral species, and negatively charged ions elute at different time intervals. The extent and speed of this separation are determined by differences in the electrophoretic mobilities of the analytes, the length of the capillary, the bulk electroosmotic flow and by the strength of electric field.
FIG. 1 is a schematic illustration of the customary arrangement of a CZE system. In this arrangement, a complete high voltage electrical circuit must be formed between opposite ends of the capillary filled with a buffer solution. This is accomplished by immersing both ends of the capillary in beakers of the buffered solutions at each end of the system.
CZE detection is currently limited to analysis by ultraviolet or fluorescent detection techniques, so as not to degrade the quality of the separation. Such detection techniques have been adequate for species that fluoresce, absorb, or are amenable to derivatization with fluorescing or absorbing chromophores. These detectors also impose cell volume and sample size limitations that preclude high separation efficiencies concurrent with high sensitivities. Structural information necessary for the correct identification of unknown analytes and their constituents cannot be obtained using these detectors due to the small sample volume and the limited spectroscopic data inherent in UV and fluorescence detection techniques. These limitations constitute a major drawback in the use of CZE for the separation and identification of complex mixtures since many compounds cannot be detected, and, if detectable, cannot be unambiguously identified. A detailed discussion of CZE can be found in an article by Jorgenson, et al., in the publication "Science" (1983), Vol. 222, beginning at page 266.
A well-known analytical technique which combines a separation technique with an analytical detection device is gas chromatography-mass spectrometry (GC-MS). In this method, GC can provide separations of sufficiently volatile compounds which are then ionized and analyzed by mass spectrometry. GC-MS has become established as the definitive analytical technique for amenable compounds, i.e., compounds having sufficient volatility for GC separation and ionization by conventional gas phase electron impact or chemical ionization methods used in mass spectrometry.
Such an established capability of broad application is not known to exist for nonvolatile compounds and mixtures. Systems for combining liquid chromatography with mass-spectroscopy are described in U.S. Pat. No. 4,209,696 and in European Patent Application 84302751.7, which are incorporated herein by reference. In these systems, carried liquid from a liquid chromatograph is electrosprayed and then analyzed by mass spectrometry. To work, electrospray requires an ionic strength of less than about 10.sup.-2 molar. Various other attempts to combine liquid chromatography with mass spectroscopy are described in "Microcolumn High Performance Liquid Chromatography, P. Kucera, Ed., J. Chromatography Library, Vol. 28, Chap. 8, pp. 260-300 (1984) and in "Small Bore Liquid Chromatography Columns: Their Properties and Uses," R.P.W. Scott, ed., Vol. 72, pp. 104-114 (1984). Unfortunately, these systems and other LC-MS approaches suffer significant limitations due to their inability to effectively separate complex mixtures, their limited separation efficiency, and the time required for analysis or separation. Combined liquid chromatography-mass spectroscopy does not provide high resolution separations. In liquid chromatography, the maximum number of theoretical plates is limited to about 10,000 for reasonable separation times (under about one hour). In contrast, CZE has been shown to be able to provide over one million theoretical plates in the same time.
Accordingly, a need remains for a method of separation that has the high-resolution separation of efficiencies of CZE and, additionally, an ability to analyze a wide range of nonvolatile compounds.